The Higher Hyaluronan In The Ectopic Milieu Contributes To Pathogenesis Of Endometriosis Via Inducing Mono-macrophage Tolerance

Endometriosis is a very frequent gynecological disorder which is characteristic of the endometrium appearance and growth outside of uterus. The incidence of this disease showed an increasing trend in recent years. As we have known that the retrograde menstruation occurs in almost cycles, only 10-15% fertile women suffer from endometriosis suggesting that the onset of endometriosis involves inefficient clearance of the menstrual efflux from the pelvis. Although it has been generally accepted that a defect of the serous immune system is involved in the pathogenesis of this disease, the underlying exact mechanisms have not been fully elucidated.Monocyte/macrophages are the first defense line in the immune response upon the foreign antigen. They are prodigious phagocytic cells that clear a large number of antigens and in turn activate the effector or/and regulatory T cells as APCs. Thus, monocytes play an important role in both innate and adaptive immunity. Chemokine is required in recruiting them from peripheral blood into peritoneal cavity, and the peritoneal macrophages may promote the onset and development of endometriosis by secreting a series of cytokines. The peritoneal macrophages isolated from patients with endometriosis have been found to have phenotypic and functional alternations leading to poor phagocytic capacity, which is highly associated with severity of endometriosis. To explore the key factors which influence the function of macrophages may provide some valuable new insights into molecular mechanisms of pathogenesis of endometriosis and a novel strategy for therapy.The endometriosis is of benign disease while some features similar to the tumor as the ectopic growth of endometrium. Hyaluronan (HA) is a major matrix constituent that normally exists as a high-molecular-weight (HMW) (>1000kDa) polysaccharide composed of repeating units of glucuronic acid and N-acetylglucosamine. Clinical and experimental studies have indicated that HA is not only a space-filling material, but also a key regulator for a series of the biological behavior, and the bioactive properties of HA vary with its size. HA is overproduced in cancer tissues, and in some cases HA level is recognized as predictive of malignancy and poor prognosis. HA not only promotes the migration of cancer cells but also inactivate the macrophages, which represents a novel mechanism for the tumor escape. Therefore, it may be speculated that HA fragments may also play an important role in pathogenesis of endometriosis.Endometriosis is an estrogen-dependent inflammatory endocrine disease. The over-expression of estrogen-regulated genes caused by the abnormal elevated level of estrogen or the declined sensitivity to progesterone contributes to development of this disease. Recently, environmental contaminants have been also suggested to play a role in pathogenesis of endometriosis. TCDD has both estrogenic and anti-estrogen effect. Research based on primates has shown that exposure to TCDD is associated with an increased prevalence and severity of endometriosis.We propose that the abnormal levels of HA in peritoneal cavity of patients with endometriosis promote the plantation and growth of reflux endometrium by regulating immunocell functions including monocytes/macrophages in the ectopic milieu.1. The level and source of HA in peritoneal cavity of patients with endometriosisThe level of HA in peritoneal cavity of patients with or without endometriosis was analyzed by ELISA. It has been found that HA level is significantly higher in peritoneal cavity with endometriosis than that of the fertile control (P<0.01), and the HA level of the III-IV stage is higher significantly than that ofⅠ～Ⅱstage of endometriosis, about 100 fold more than that of the control. There is a positive correlation of HA level to stage of the disease.To explore source of the high level of HA, we separated the ESCs from eutopic and ectopic tissues of endometriosis with normal fertile endometrium as control. The ESCs were stimulated by different concentration of estrogen, progesterone (10-10～10-7M) and TCDD (0.01～10nM), respectively, and HA level in the supernatant was assayed by ELISA. The results show that macrophages secret low level of HA that is unaffected by exogenous hormones and TCDD. HA secretion of eutopic ESCs is higher significantly than that of ectopic ESCs (P＜0.05), and HA secretion of both is more than that of the control (P＜0.01). Estrogen alone shows a visible effect on HA secretion of the ESCs of the three different sources, and Progesterone alone can also stimulate HA secretion of the ESCs, but inhibits the estrogen-induced increase of HA secretion, especially in the normal ESCs. We have found that TCDD can enhance HA secretion of ESCs, especially the eutopic ESCs, in a dose-dependent manner. TCDD presents anti-estrogen effect in the normal ESCs while estrogenic effect in the eutopic and ectopic ESCs on HA secretion.Among the three HA synthase (HAS) isoforms, HAS2 shows the strongest activity mainly involved in HMW-HA synthesis, and has been considered as the most important synthase for the HA production. To investigate whether HAS2 in ESCs also can be affected by steroid hormone and TCDD, we performed a series of in cell western assay. The results show that estrogen, progesterone and TCDD alone can stimulate the expression of HAS2 in all the three kinds of ESCs. Progesterone can inhibit the up-regulation of estrogen on HAS2 expression, especially in the normal ESCs. TCDD presents anti-estrogen effect in the normal ESCs while estrogenic effect in the eutopic and ectopic ESCs on HAS2 expression.In summary, we have found that there is very higher level of HA in peritoneal cavity of patients with endometriosis that is produced by the eutopic and ectopic ESCs. The secretion of HA is regulated by the female steroid hormone and environmental pollutant TCDD, which suggests that the higher level of HA in the ectopic milieu may play an important role in pathogenesis of endometriosis. 2. HA of the different molecular weight induces formation of the immunotolerant macrophages and inhibits apoptosis of these cellsIn light of the results above, we performed several sets of experiments to investigate role of HA in the immunotolerant polarization of macrophages. Exposure to HMW-HA and MMW-HA drives monocytes to develop into the tolerant macrophages that display an increased CD 14 expression and decreased HLA-DR and CD86 expression. Although LMW-HA and ULMW-HA had little or no impact at any concentration, they can up-regulate expression of CD44 on monocytes.We have also found that all HA of the four different molecular weight can significantly inhibit apoptosis of the macrophages, which suggests that HA can improve survival of the tolerant monocytes.3. The eutopic and ectopic ESCs-derived HA induces formation of the immunotolerant macrophages and up-regulates their tolerant functionWe constructed three ESCs-monocytes co-culture in vitro to elucidate effect of ESCs and role of HA on monocytes. The monocytes in co-culture with the eutopic or ectopic ESCs had a markedly altered phenotype showing an increased expression of CD14 and decreased expression of HLA-DR andCD86, and interestingly, CD44, the receptor of HA, increases significantly. The immunotolerant phenotype of monocytes cannot then response appropriately upon LPS stimulation. We next determined the effect of CD44 mAb on the ESCs-induced monocytes dysfunction. The results show that pretreatment of monocytes with anti-CD44 antibody can effectively inhibit the alternation of monocyte phenotype. The HA-specific antagonist peptide (Pep-1) can also inhibit markedly the formation of immunotolerant monocytes.The macrophages can secrete higher level of IL-10 than the three kinds of ESCs. The eutopic and ectopic ESCs secrete higher level of IL-10 than that of the normal ESCs. The respective contact co-culture of eutopic or ectopic ESCs with macrophages shows a significant up-regulation of IL-10 secretion, and Pep-1 remarkably inhibits the enhanced secretion of IL-10 in the co-culture, which suggests that HA in the co-culture may up-regulate the production of IL-10 by macrophages. The macrophages also can secrete higher level of IL-12p70 than the three kinds of ESCs. The eutopic and ectopic ESCs produce higher level of IL-12p70 than that of the normal ESCs. The respective contact co-culture of eutopic or ectopic ESCs with macrophages can decrease the secretion of IL-12p70, and Pep-1 remarkably reverse the decreased secretion of IL-12p70 in the co-culture, which suggests that HA in the co-culture may also down-regulate the production of IL-12p70 by macrophages.In conclusion, it has been demonstrated in the present study that there presents a very higher level of HA in peritoneal cavity with endometriosis that is produced by the eutopic and ectopic ESCs. Estrogen, progesterone and TCDD are involved in regulation of HA production via regulating HA synthase expression. The larger HA can induce the formation of immunotolerant macrophages with increased secretion of IL-10 and decreased expression of IL-12. The HA-mediated declined apoptosis further leads to survival and functions of the tolerant monocytes in the progress of this disease. Our research uncovers a possible new molecular mechanism of monocytes tolerance in pathogenesis of endometriosis.